RED crucian carp (Carassius auratus red var., $, 2n ¼

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1 Copyright Ó 2006 by the Genetics Society of America DOI: /genetics Evidence for Recombination of Mitochondrial DNA in Triploid Crucian Carp Xinhong Guo, Shaojun Liu 1 and Yun Liu College of Life Sciences, Hunan Normal University, ChangSha , Hunan, People s Republic of China Manuscript received August 21, 2005 Accepted for publication November 14, 2005 ABSTRACT In this study, we report the complete mitochondrial DNA (mtdna) sequences of the allotetraploid and triploid crucian carp and compare the complete mtdna sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtdna nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtdna fragment (12,759 bp) derived from the paternal fish. These results suggest that mtdna recombination was derived from the fusion of the maternal and paternal mtdnas. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtdna fuse with the mtdna of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtdna recombination. In summary, our results provide the first evidence of mtdna combination in polyploid fish. RED crucian carp (Carassius auratus red var., $, 2n ¼ 100) and common carp (Cyprinus carpio L., #,2n ¼ 100) belong to different genera. The crossing between them is considered distal. The F 2 hybrids were derived from mating males with females of F 1, and F 3 hybrids were produced by mating males with females of F 2. The cytological analysis revealed that the females and males of F 2 hybrids were able to produce diploid eggs and diploid sperms that fertilized each other to form tetraploid fish in F 3. Thus, the hybrids of F 3 F 11 of red crucian carp and common carp were proved to be allotetraploids (4n ¼ 200) with two chromosome sets of red crucian carp and two chromosome sets of common carp (Liu et al. 2001; Sun et al. 2003). In the allotetraploid population, both females and males were fertile. Similar to F 3 F 11,F 12 F 14 hybrids were also confirmed as allotetraploids (our unpublished data). The shape of the F 1 F 2 was intermediate to red crucian carp and common carp. The tetraploids of F 3 F 14 generations displayed a similar morphological phenotype (shape and color) also intermediate to red crucian carp and common carp, which could be stably inherited from generation to generation. The phenotype of F 3 F 14 hybrids was a little different from that of F 1 F 2. Thus, 1 Corresponding author: College of Life Sciences, Hunan Normal University, Changsha , Hunan, People s Republic of China. lsj@hunnu.edu.cn our previous work established an allotetraploid population in the vertebrate through successive generations of hybridization. Our successful establishment of the allotetraploid population in the vertebrate provided important diploid-gamete resources to create the sterile triploid crucian carps through mating F 3 F 14 hybrids (#) with Japanese crucian carps (C. auratus cuvieri) ($). The triploid crucian carps are sterile, faster in growth rate, and have a higher survival rate and thus greatly enhance their annual production. The triploid crucian carps have been produced on a large scale and have provided an important source of white meat in China. During recent years,.100,000 tetraploids and.300 million triploids were annually produced in China. As a new species with 200 chromosomes, the tetraploid population (F 3 F 14 ) showed very important significance in the application due to the generation of the sterile triploids by interploidy crossing between tetraploids and diploids. Thus, it is our interest to reveal the genetic relationships between triploid crucian carp and its female parent Japanese crucian carp and its male parent allotetraploid in the mitochondrial DNA (mtdna) structures. The mtdna of most animals is a 16- to 20-kbp, circular genome essential for the maintenance of mitochondrial function and is present in multiple copies in most cell types. High sequence divergence and maternal inheritance make mtdna useful in tracing animal Genetics 172: (March 2006)

2 1746 X. Guo, S. Liu and Y. Liu lineages. Whether recombination occurs between mitochondrial genomes is a longstanding question in mitochondrial biology, animal evolution, and population studies. The paternal inheritance of mtdna, that is, transmission of the paternal mtdna into the egg and its survival in the adult organism, was demonstrated in a number of species (Kondo et al. 1990; Gyllensten et al. 1991; Magoulas and Zouros 1993; Skibinski et al. 1994; Zouros et al. 1994; Schwartz and Vissing 2002; Kvist et al. 2003). Moreover, direct evidence of recombination now existed in four animal species: the nematode Meloidogyne javanica (Lunt and Hyman 1997), mussel sister-species Mytilus galloprovincialis (Ladoukakis and Zouros 2001) and M. trossulus (Burzynski et al. 2003), flatfish Platichthys flesus (Hoarau et al. 2002), and human (Kraytsberg et al. 2004). In this study, we provided the first direct evidence of mtdna recombination in a polyploid cyprinid, triploid crucian carp. MATERIALS AND METHODS Animals: The five allotetraploids, 5 Japanese crucian carps, and 10 triploid crucian carps were collected from the Chinese national tetraploid fish protection station located at Hunan Normal University. Preparation of total DNA: Total DNAs were extracted individually from blood samples of five allotetraploids, 5 Japanese crucian carps, and 10 triploid crucian carps using a DNA extraction kit from Shanghai Sangon. Isolation and sequencing of the COIII gene: The COIII genes of five allotetraploids, 5 Japanese crucian carps, and 10 triploid crucian carps were amplified, respectively, by PCR using the following primers: mtdna13l (59-GTCCGACTCACA GCCAACT-39) andmtdna13h(59-gtgggagtcagaaagaa ACG-39). The PCR fragments were then cloned into T-vector and sequenced on both strands with the ABI 377 automatic sequencer. Isolation and sequencing of the mitochondrial genome: The highly conserved and newly designed PCR primers in the online supplemental Table 1 ( supplemental/) were used to amplify up to 20 contiguous and overlapping fragments of the mitochondrial molecules in allotetraploid and triploid crucian carp. The PCR reactions were carried out in a 50-ml final volume containing 200 ng DNA, 1.8 mmol MgCl 2, 0.2 mmol of each dntp, 0.4 mmol of each primer, 13 Ex Taq buffer, and 1.25 units of TaKaRa (Berkeley, CA) Ex Taq. The protocol for amplifications was 94 for 120 sec and 30 cycles at 94 for 60 sec, for 60 sec, and 72 for sec. A majority of PCR products were directly sequenced, and some fragments that were difficult to be sequenced using PCR products were cloned into T-vector and then sequenced. Sequencing reactions were performed using the Big Dye Terminator cycle sequencing kit version 2.0 and run with the ABI 377 automatic sequencer. The mitochondrial (mt) genome sequences of the allotetraploid and triploid crucian carp have been submitted to GenBank under accession nos. AY and AY771781, respectively. Sequence identity: All sequences were analyzed using the Blast ( CLUSTALW ( and MEGA 1.0 (Kumar et al. 1993) programs to determine the identity. The mt genome sequence of Japanese crucian carp (AB045144) was retrieved from the GenBank database. RESULTS COIII gene: The mt COIII gene sequences of 786 bp were obtained from allotetraploids, Japanese crucian carps, and triploid crucian carps after removing areas of ambiguity as well as other data. The five allotetraploids and 5 Japanese crucian carps shared one haplotype, respectively. The haplotype sequence of Japanese crucian carp was the same as that reported by the GenBank database. However, 10 triploid crucian carps shared the two different haplotypes (TC-1 and TC-2, respectively). Among the 10 tested triploid crucian carps, only 1 individual shared the TC-1 haplotype, and the other 9 individuals shared the TC-2 haplotype. The nucleotide identity (99.1%) between the haplotype TC-2 of triploid crucian carps and its female parent Japanese crucian carp was higher than that (95%) between the triploid crucian carp and its male parent allotetraploid. However, the nucleotide identity (95.5%) between the haplotype TC-1 of triploid crucian carps and its female parent Japanese crucian carp was lower than that (100%) between the triploid crucian carp and its male parent allotetraploid. Such a result strongly showed the mtdna paternal inheritance characteristic in the COIII gene of the haplotype TC-1 of triploid crucian carps. Identity analysis: To verify whether the paternal inheritance of the COIII gene was the recombination result of the maternal and paternal mtdna in triploid crucian carp, we chose the haplotype TC-1 of triploid crucian carps and one allotetraploid to obtain the complete mtdna sequence and observed the genetic relationship of the mt genomes between triploid crucian carp and its parents. The percentages of nucleotide identities of the separate regions of the mt genomes in the triploid crucian carp and its parents are shown in Table 1. The complete mtdna nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. The result indicated that the obvious paternal inheritance characteristic was in the triploid crucian carp mt genome. In addition, from trna-val to ND5 (12,759 bp), the separate gene nucleotide identity between the triploid crucian carp and its male parent allotetraploid was higher than or equal to that between the triploid crucian carp and its female parent Japanese crucian carp. However, between the following regions (NADH 6 / trna-gly / Cytb / trna-thr / trna- Pro / Control region / trna-phe / 12s rrna), the nucleotide identity between the triploid crucian carp and its male parent allotetraploid was lower than or equal to that between the triploid crucian carp and its female parent Japanese crucian carp (Table 1). That is to say, a pattern of identity and difference existed at synonymous sites of mitochondrial genomes between triploid crucian carp and its parents. The results indicated that the

3 Mitochondrial DNA Recombination of Triploid Crucian Carp 1747 TABLE 1 Percentage of nucleotide identities of separate regions of mitochondrial genomes in triploid crucian carp (TC), its female parent Japanese crucian carp ( JC), and its male parent allotetraploid (AT) Region TC and JC TC and AT JC and AT Complete mtdna Control region trna-phe s rrna trna-val s rrna trna-leu NADH trna-ile trna-gln (L) trna-met NADH trna-trp trna-ala (L) trna-asn (L) trna-cys (L) trna-tyr (L) COI trna-ser trna-asp COII trna-lys ATPase ATPase COIII trna-gly NADH trna-arg NADH 4L NADH trna-his trna-ser trna-leu NADH NADH 6 (L) trna-gly(l) Cytb trna-thr trna-pro (L) (L), a gene encoded by the L-strand. recombination of the maternal and paternal mtdna occurred in the development of the triploid crucian carp. DISCUSSION In this study, we provided evidence of paternal inheritance, using the COIII gene as a marker. Our results revealed that the COIII nucleotide identity (100%) between the haplotype TC-1 of triploid crucian carps and its male parent allotetraploid was higher than that (95.5%) between the haplotype TC-1 of triploid crucian carps and its female parent Japanese crucian carp, thus providing the first example of the paternal inheritance characteristic of mtdna in polyploid fish. In fact, Ladoukakis and Zouros (2001) provided strong evidence for mtdna recombination in the COIII gene in gonadal tissue in the marine mussel M. galloprovincialis from the Black Sea. However, the mussel system was an exception, for which two mtdna lineages existed, one transmitted through the egg (the F lineage) and the other transmitted through the sperm (the M lineage) (Zouros et al. 1994). The comparative study on sequence identities of the mt genomes between triploid crucian carp and its parents provided direct evidence of mtdna recombination. Because recombination was considered as an indispensable part of DNA replication and repair (Kowalczykowski 2000), the elevated mtdna mutation rate in animals, compared with that of nuclear DNA (Wallace et al. 1987), was taken as an indication of absence of homologous recombination in animal mtdna (Howell 1997). It may be possible that the mtdna of triploid crucian carp was first inherited from the female parent and then through mutation displayed the high nucleotide identity (98%) with its male parent. However, considering the differences between the triploid crucian carp and its female parent Japanese crucian carp, it would need many point mutations of the long sequence from trna- Val to ND5 (12,759 bp) to obtain the observed identity. In reality, many mtdna positions in the closely related cyprinids were highly conserved, and thus such frequent point mutation was unlikely. Another possibility generating the observed nucleotide identity was the presence of a PCR artifact because Taq polymerase could produce chimeric DNA molecules (i.e.,pcrjumpingsensu Paabo et al. 1990) when damaged fragments of a mixed template of DNAs were joined during the amplification process. This was also impossible for two reasons. First, Ladoukakis and Zouros (2001) provided direct evidence against artifacts by cloning of PCR product molecules from artificially heteroplasmic targets. Second, the DNA samples used in this research were observed to be homoplasmic because each reaction with an individual sample produced only a single PCR product. The mtdna recombination observed in triploid crucian carp seemed to be different from other mtdna recombination found in the nematode M. javanica (Lunt and Hyman 1997) and various other animal species (Solignac et al. 1986; Snyder et al. 1987; Rand and Harrison 1989; Buroker et al. 1990; Ludwig et al. 2000). The latter type of mtdna recombination was mediated by a mechanism of unequal crossing over that resulted in products of unequal lengths of mtdna. It was restricted to parts of the genome where tandem repeats occurred. For the majority of animal mtdna, such arrays of repeats occurred in the replication control region. Our observations referring to the complete mt genome implied that homologous recombination could occur in any part of the triploid crucian carp mt

4 1748 X. Guo, S. Liu and Y. Liu genome. The mtdna recombination found in our study was also anticipated by Thyagarajan et al. (1996), who observed that mitochondria in cultured human cells contained the enzymes that catalyzed nuclear recombination. The implication, therefore, was that these enzymes were imported in the mitochondrion as well as in the nucleus and that the molecular mechanism of mtdna recombination was similar to that of nuclear recombination. Recent studies by Maynard Smith and Smith (2002) also provided overwhelming evidence for regions of identity and difference between mtdna sequences for synonymous sites, which was explained by recombination. More recently, the mtdna recombinants in muscle tissue of a man with paternal inheritance of the mitochondrial genome were observed, and it was suggested that the enzymes that were responsible for replicating mtdna stopped replicating maternal mtdna and jumped to the corresponding paternal mtdna position to replicate paternal mtdna (Kraytsberg et al. 2004). Furthermore, Kraytsberg et al. (2004) assumed that the mtdna recombinants fell into two structural classes: class 1, with a short paternal sequence inserted into a mostly maternal molecule, and class 2, with a maternal sequence flanked by paternal sequences. In our study, the mtdna recombinant in the triploid crucian carp may belong to the structural class 2 because its maternal mtdna sequence (NADH 6 / trna-gly / Cytb / trna-thr / trna-pro / Control region / trna-phe / 12s rrna) was surrounded by the longer paternal mtdna sequence (from trna-val to ND5). Regardless, our demonstration of the presence of identity and difference at synonymous sites of mitochondrial genomes between triploid crucian carp and its parents provided clear evidence for the mtdna recombination. Triploid crucian carp of allotetraploid 3 Japanese crucian carp proved to be allotriploid (3n ¼ 100) with two chromosome sets of allotetraploids and one chromosome set of Japanese crucian carp (Liu et al. 2001). We reasoned that the mtdna recombination of the triploid crucian carp resulted from the fusion of the maternal mtdna and paternal leakage mtdna. Compared with the haploid egg with one set of the genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily effect to make its mtdna leak and then fuse with the mtdna of the haploid egg. In addition, the triple hybrid nature of the crucian carp could make it relatively easy to form the mtdna recombination. The mtdna recombination we revealed through the sequence identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents has several implications for vertebrate mtdna in general. Prior to the occurrence of the mtdna recombination, the maternal and paternal mitochondria should be present in the same cell. This implied that paternal mtdna leakage was followed by fusion of the mitochondria. Paternal leakage was reported for mice (Gyllensten et al. 1991), anchovies (Magoulas and Zouros 1993), and great tit (Kvist et al. 2003). For mice, the leakage of paternal mtdna was estimated at 10 ÿ4 of an individual s mtdna pool. These observations suggested that incidental paternal mtdna transmission could be the rule in animals, despite the presence of mechanisms for sperm mtdna elimination in the fertilized ovum (Shitara et al. 1998; Sutovsky et al. 1999). The fusion of mitochondria was also demonstrated in Drosophila (Yaffe 1999), and the enzymes necessary for recombination were found in human mitochondria (Thyagarajan et al. 1996). It appeared therefore that all these properties were present in triploid crucian carp and probably in vertebrates. In previous studies, direct evidence for the animal mtdna recombination was provided in the nematode M. javanica (Lunt and Hyman 1997), mussel sisterspecies M. galloprovincialis (Ladoukakis and Zouros 2001) and M. trossulus (Burzynski et al. 2003), flatfish P. flesus (Hoarau et al. 2002), and human (Kraytsberg et al. 2004). In this study, mtdna recombination was presented in triploid crucian carp. Our results provided the first report of mtdna recombination in the polyploid cyprinid. The importance of recombination in vertebrate mitochondria has broad implications across several fields, ranging from human mitochondrial diseases (Schon 2000) to the compromise of phylogenetic and population studies that assumed strict clonal inheritance of mtdna (Schierup and Hein 2000). In the case of human mitochondrial diseases, mtdna recombination will greatly change the modes and patterns of inheritance, which in turn may affect current diagnostic methods. If homologous recombination occurs in animal mtdna, it will have an important effect on our understanding of mtdna mutation and repair mechanisms and rates of mutation accumulation. Homologous recombination was essential for DNA repair in yeast (Ling et al. 1995) and was expected to play a similar role in animal mtdna (Thyagarajan et al. 1996; Howell 1997). Recombination errors may lead to unequal crossing over and deletions between short direct repeats of the type associated with mitochondrial diseases (Holt et al. 1988). It is essential to emphasize the implications of homologous recombination of mtdna for mitochondrial disease caused by mtdna deletions and mutations. Recombination can also affect the accuracy of phylogenetic reconstruction (Posada and Crandall 2002), inferences related to demographic history, and the application of molecular clocks (Schierup and Hein 2000). Especially, with regard to the use of mtdna for evolutionary studies, it would mean that we should not, as it has been common practice until now, draw conclusions about the evolutionary history of the entire mitochondrial genome by looking at parts of it. Such extrapolations are, for example, implicit in studies of hybridization and introgression in natural populations (Ferris et al. 1983; Powell

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